The role of the insulin receptor C-terminal domain in the regulation of insulin signal transduction was studied with a synthetic peptide (peptide HC) whose structure corresponds to residues 1293-1307 of the insulin proreceptor sequence. Peptide HC enhanced insulin-stimulated autophosphorylation of the insulin receptor in cell-free systems and in digitonin-permeabilized cells without any detectable effect on basal authophosphorylation levels on receptor dephosphorylation. The peptide was modified by addition of a stearyl moieties at its N-terminus, and introduced in intact Chinese hamster ovary (CHO) cells transfected with an expression plasmid encoding the human insulin receptor. We found that stearyl-peptide HC enhanced several fold insulin-stimulated insulin receptor autophosphorylation while having no effect on ligand-stimulated receptor phosphorylation activity in CHO cells overexpressing either the IGF-1 receptor or EGF receptor. To explore the effect of stearyl-peptide HC in regulating insulin signaling, we evaluated the association of phosphatidylinositol 3'-kinase (PI e'-K) to tyrosine phosphorylated insulin receptor substrate-1 (IRS-1), and the levels of phosphorylation of mitogen-activated protein (MAP) kinase by hyperphosphorylation gel shift assay. Loading of cells with stearyl-peptide HC resulted in a 1.7-fold increase in the amount of insulin-stimulated PI 3'-K activity detected in anti-IRS-1 immunoprecipitates, and a 1.8-fold increase in MAP kinase hyperphosphorylated species. Taken together our data provide evidence for the important role of the sequence 1293-1307 in the C-terminus of the insulin receptor in the transmission of biological effects and could account, as least in part, for receptor specificity.